luminal-A Breast Cancer Research Articles

Autocrine phosphatase PDP2 inhibits ferroptosis by dephosphorylating ACSL4 in the Luminal A Breast Cancer.

Phosphatases can dephosphorylate phosphorylated kinases, leading to their inactivation, and ferroptosis is a type of cell death. Therefore, our aim is to identify phosphatases associated with ferroptosis by analyzing the differentially expressed genes (DEGs) of the Luminal A Breast Cancer (LumABC) cohort from the Cancer Genome Atlas (TCGA). An analysis of 260 phosphatase genes from the GeneCard database revealed that out of the 28 DEGs with high expression, only the expression of pyruvate dehydrogenase phosphatase 2 (PDP2) had a significant correlation with patient survival. In addition, an analysis of DEGs using gene ontology, Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analysis revealed a significant variation in the expression of ferroptosis-related genes. To further investigate this, we analyzed 34 ferroptosis-related genes from the TCGA-LumABC cohort. The expression of long-chain acyl-CoA synthetase 4 (ACSL4) was found to have the highest correlation with the expression of PDP2, and its expression was also inversely proportional to the survival rate of patients. Western blot experiments using the MCF-7 cell line showed that the phosphorylation level of ACSL4 was significantly lower in cells transfected with the HA-PDP2 plasmid, and ferroptosis was correspondingly reduced (p < 0.001), as indicated by data from flow cytometry detection of membrane-permeability cell death stained with 7-aminoactinomycin, lipid peroxidation, and Fe2+. Immunoprecipitation experiments further revealed that the phosphorylation level of ACSL4 was only significantly reduced in cells where PDP2 and ACSL4 co-precipitated. These findings suggest that PDP2 may act as a phosphatase to dephosphorylate and inhibit the activity of ACSL4, which had been phosphorylated and activated in LumABC cells. Further experiments are needed to confirm the molecular mechanism of PDP2 inhibiting ferroptosis.

Cholesterol biogenesis is a PTEN-dependent actionable node for the treatment of endocrine therapy-refractory cancers.

PTEN and PIK3CA mutations are the most prevalent PI3K pathway alterations in prostate, breast, colorectal, and endometrial cancers. p110β becomes the prominent PI3K isoform upon PTEN loss. In this study, we aimed to understand the molecular mechanisms of PI3K dependence in the absence of PTEN. Using online bioinformatical tools, we examined two publicly available microarray datasets with aberrant PI3K activation. We found that the rate-limiting enzyme of cholesterol biogenesis, SQLE, was significantly upregulated in p110β-hyperactivated or PTEN-deficient mouse prostate tumors. Concomitantly, the expression of cholesterol biosynthesis pathway enzymes was directly correlated with PI3K activation status in microarray datasets and diminished upon PTEN re-expression in PTEN-null prostate cancer cells. Particularly, PTEN re-expression decreased SQLE protein levels in PTEN-deficient prostate cancer cells. We performed targeted metabolomics and detected reduced levels of cholesteryl esters as well as free cholesterol upon PTEN re-expression. Notably, PTEN-null prostate and breast cancer cell lines were more sensitive to pharmacological intervention with the cholesterol pathway than PTEN-replete cancer cells. Since steroid hormones use sterols as structural precursors, we studied whether cholesterol biosynthesis may be a metabolic vulnerability that enhances antihormone therapy in PTEN-null castration-resistant prostate cancer cells. Coinhibition of cholesterol biosynthesis and the androgen receptor enhanced their sensitivity. Moreover, PTEN suppression in endocrine therapy-resistant luminal-A breast cancer cells leads to an increase in SQLE expression and a corresponding sensitization to the inhibition of cholesterol synthesis. According to our data, targeting cholesterol biosynthesis in combination with the hormone receptor signaling axis can potentially treat hormone-resistant prostate and breast cancers.

Identifying prognostic subgroups of luminal-A breast cancer using deep autoencoders and gene expressions.

Luminal-A breast cancer is the most frequently occurring subtype which is characterized by high expression levels of hormone receptors. However, some luminal-A breast cancer patients suffer from intrinsic and/or acquired resistance to endocrine therapies which are considered as first-line treatments for luminal-A breast cancer. This heterogeneity within luminal-A breast cancer has required a more precise stratification method. Hence, our study aims to identify prognostic subgroups of luminal-A breast cancer. In this study, we discovered two prognostic subgroups of luminal-A breast cancer (BPS-LumA and WPS-LumA) using deep autoencoders and gene expressions. The deep autoencoders were trained using gene expression profiles of 679 luminal-A breast cancer samples in the METABRIC dataset. Then, latent features of each samples generated from the deep autoencoders were used for K-Means clustering to divide the samples into two subgroups, and Kaplan-Meier survival analysis was performed to compare prognosis (recurrence-free survival) between them. As a result, the prognosis between the two subgroups were significantly different (p-value = 5.82E-05; log-rank test). This prognostic difference between two subgroups was validated using gene expression profiles of 415 luminal-A breast cancer samples in the TCGA BRCA dataset (p-value = 0.004; log-rank test). Notably, the latent features were superior to the gene expression profiles and traditional dimensionality reduction method in terms of discovering the prognostic subgroups. Lastly, we discovered that ribosome-related biological functions could be potentially associated with the prognostic difference between them using differentially expressed genes and co-expression network analysis. Our stratification method can be contributed to understanding a complexity of luminal-A breast cancer and providing a personalized medicine.

Comparative evaluation of the therapeutic strategies using a minimal model of luminal-A breast cancer

Cellular behavior is heavily influenced by cellular interactions, which are often lost in conventional cell culture methods. As a result, in vitro cellular behavior may not accurately reflect in vivo conditions. Three-dimensional (3D) culture, on the other hand, is better suited for studying cellular behavior as it allows for more comprehensive cell communication. In this study, we utilized 3D culture of the MCF-7 cell line to create a minimal model of luminal-A breast cancer and evaluated its histopathological and morphological features using various methods.To determine the optimal therapeutic strategies for eliminating cancer cells, we assessed the effectiveness of diverse therapeutic approaches, including targeting distinct phases of the cell cycle, endocrine therapy, and gene therapy in both 2D and 3D culture systems. Our findings indicate that cells derived from mammospheres respond differently to their parent cells in monolayer culture depending on the therapeutic strategy used. This variability in drug response may be due to the altered microenvironment created by heterogeneous cellular makeup and emerging cellular interactions in the 3D culture. Therefore, it is important to administer a therapeutic approach that can eradicate cells regardless of the microenvironment.

Breast Tumor Cells Evade the Cytotoxic Action of Anastrozole, Aspirin, and Clopidogrel Cocktail.

Globally, breast cancer is among the most frequently diagnosed and common cause of death among women. Aromatase inhibitors, such as anastrozole, are one of the first-line therapies used in the treatment of breast cancer in postmenopausal women; however, thromboembolic complications are common. Thus, this study investigated the combined effects of anastrozole and antiplatelet therapies, aspirin and clopidogrel, on breast cancer cytotoxicity and survival in vitro. Breast cancer cell lines (MCF-7 and T47D) were treated with varying Cmax concentrations of anastrozole and/or antiplatelet therapies for 24 h. A wound-healing scratch assay was used to measure migration and the WST-1 assay for cellular proliferation. An autophagy/cytotoxicity dual staining kit was used to assay cell death and survival. Changes in cell morphology were assessed using scanning electron microscopy. Data were analyzed with Statistica software. Our findings showed that sub-phenotypic differences exist between the luminal-A breast cancer cell lines, with T47D cells being more aggressive than MCF-7 cells. Cellular proliferation and migration responded in a dose-dependent manner for the different treatment groups. Notably, anastrozole combined with aspirin and clopidogrel mediated higher levels of cell survival than each agent individually, with autophagy levels being significantly increased in comparison to that induced with antiplatelet therapy alone.

Breast cancer cells interact with tumor-derived extracellular matrix in a molecular subtype-specific manner

Mimicking the native microenvironment is vital for tumor engineering. Breast cancer is a highly heterogeneous disease with various molecular subtypes exhibiting distinct biological behaviors and treatment responsiveness. The heterogeneity of extracellular matrix (ECM) of breast cancer has remained largely unexplored and underestimated. The present study addressed this issue by comparing the composition, architecture, and functional roles of ECMs derived from breast cancers of two molecular subtypes, which are luminal-A breast cancer (less aggressive, ERα+)-derived ECM (LA-ECM) and triple-negative breast cancer (high aggressive, ERα-)-derived ECM (TN-ECM). Compared with normal breast tissue-derived ECMs (B-ECM), tumor-derived ECMs showed higher contents of pro-collagen I, fibronectin, and laminin, in addition with a significantly altered architecture. Transcriptome sequencing revealed that, compared with those cultured with B-ECM, MCF7 cells (an estrogen receptor (ER)α + luminal-A breast cancer cell line) cultured with LA-ECM and TN-ECM showed approximately 9.65 % and 9.04 % changes in the expression of all detected genes, respectively. The TN-ECM induced proliferation, promoted epithelial-to-mesenchymal transition, downregulated ERα expression, and reduced endocrine treatment sensitivity of MCF7. Above results have elucidated the role of phenotype-specific tumor ECM in cell phenotype maintenance, treatment sensitivity, and cancer progression, which highlighted the importance of ECM heterogeneity as well as its role in tumor microenvironment engineering and drug screening.

Anticancer effect of Illicium verum (star anise fruit) against human breast cancer MCF-7 cell line.

To investigate the anticancer effect of Illicium verum against human breast cancer MCF-7 cell line. An experimental study was conducted in Multidisciplinary and Tissue Culture Laboratory, Aga Khan University in collaboration with Pharmacology Department of Bahria University Medical and Dental College, Karachi, Pakistan from January 2021 to June 2021. MCF-7 cells of Luminal-A breast cancer were seeded in 96-well plate and treated with I.verum methanol extract. After incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye was used for cell viability and cell proliferation assays to determine the number of dead and viable cells, and the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) plate reader. In cell viability assay, different doses of I. verum methanol extract were used to treat the MCF-7 (0.25, 0.5, 1, 3, 6, 12, 25, and 50μg/ml) cells. For apoptosis analysis, the cells were processed with 4´, 6-diamidino-2-phenylindole fluorescent nuclear dye (DAPI) and were examined for fluorescence intensity and apoptotic cells. For cell proliferation assay and apoptosis the IC50 dose of 5.5μg/ml I. verum methanol extract was used. The MCF-7 cells showed a significant reduction (p-value <0.01) in cell viability in the presence of all tested doses of I. verum methanol extract, except for the dose of 0.25μg/ml. The IC50 dose 5.5μg/ml of same extract also showed a significant reduction (p-value <0.01) in cell proliferation and apoptosis induction in MCF-7 cells. Illicium verum methanol extract possesses very potent anticancer action against MCF-7 cells through cytotoxicity, reduction, and inhibition of cancer cells and by inducing apoptosis.

Tumor Cell-Autonomous Pro-Metastatic Activities of PD-L1 in Human Breast Cancer Are Mediated by PD-L1-S283 and Chemokine Axes.

Simple SummaryTriple-negative breast cancer (TNBC) is an aggressive disease that responds in a limited manner to immune checkpoint blockades targeting the PD-L1/PD-1 axis, suggesting that PD-L1 potentiates TNBC progression via pathways not related to immune suppression. We demonstrated that, in human breast cancer cells, PD-L1 expression increased in a cell-autonomous manner tumor cell growth, invasion and release of pro-metastatic factors; these activities were elevated by exposure to PD-1 and were markedly impaired in S283-mutated PD-L1-expressing cells. Invasion of WT-PD-L1-expressing TNBC cells depended on autocrine chemokine circuits, involving CXCR1/2, CCR2, CCR5 and their ligands. In T cell-deficient mice, WT-PD-L1 exhibited increased tumor growth and metastasis by TNBC cells, whereas S283A-PD-L1-expressing cells showed a very poor tumorigenic and metastatic profile. These findings on cell-autonomous and PD-1-induced pro-metastatic activities of PD-L1 in cancer cells suggest that treatments targeting PD-L1 could improve the efficacy of immune-targeting checkpoint inhibitors, e.g., anti-PD-1 or anti-CTLA-4 in TNBC.Therapies targeting the PD-L1/PD-1 axis have recently been introduced to triple-negative breast cancer (TNBC) with limited efficacy, suggesting that this axis promotes tumor progression through mechanisms other than immune suppression. Here, we over-expressed WT-PD-L1 in human TNBC cells (express endogenous PD-L1) and in luminal-A breast cancer cells (no endogenous PD-L1 expression) and demonstrated that cell-autonomous PD-L1 activities lead to increased tumor cell growth, invasion and release of pro-metastatic factors (CXCL8, sICAM-1, GM-CSF). These activities were promoted by PD-1 and were inhibited by mutating S283 in PD-L1. Invasion of WT-PD-L1-cells required signaling by chemokine receptors CXCR1/2, CCR2 and CCR5 through autocrine circuits involving CXCL8, CCL2 and CCL5. Studies with T cell-deficient mice demonstrated that cell-autonomous WT-PD-L1 activities in TNBC cells increased tumor growth and metastasis compared to knock-out (KO)-PD-L1-cells, whereas S283A-PD-L1-expressing cells had minimal ability to form tumors and did not metastasize. Overall, our findings reveal autonomous and PD-1-induced tumor-promoting activities of PD-L1 that depend on S283 and on chemokine circuits. These results suggest that TNBC patients whose tumors express PD-L1 could benefit from therapies that prevent immune suppression by targeting PD-1/CTLA-4, alongside with antibodies to PD-L1, which would allow maximal impact by mainly targeting the cancer cells.

Abstract PD9-07: Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer

Abstract Background: Luminal, i.e., estrogen receptor (ER)-positive breast cancer (BC) is a heterogeneous disease in terms of tumor progression, therapy response, and relapse. Additional biomarkers with a prognostic and predictive impact would facilitate advanced patient stratification and can reveal advanced therapeutic options for individual patients suffering from luminal BC subtype. First objective of this study was the hypothesis driven validation and identification of biomarkers associated with successful engraftment, augmented tumor growth, and enhanced metastasis upon xenotransplantation into HTM and non-humanized tumor mice. Second objective was the retrospective validation and correlation of aforementioned markers with clinical outcomes (disease free-survival [DFS] and overall survival [OS]) of luminal BC patients after neoadjuvant chemotherapy within the GeparTrio trial. Methods: Immunodeficient NSG mice with and without immunological humanization were transplanted with primary, ER-positive BC tissues and cells. Engraftment rates, tumor progression, and metastasis were monitored as a function of marker expression and genomic alterations, considered to be associated with tumor cell stemness and tumor initiating capacity. Functional assays were applied to demonstrate the impact of identified markers on tumor cell viability, growth and migration in-vitro. Dual color fluorescence-in-situ-hybridization to monitor mdm2 gene and the cen12 region was applied to 502 pretherapeutic ER-positive tissue specimens of BC patients treated with anthracycline/taxane based neoadjuvant chemotherapy within the GeparTrio trial. Mdm2 gene expression was analyzed in the entire luminal BC cohort as well as in luminal-A and luminal-B samples classified based on a Ki-67 cut-off of 20% (St. Gallen guideline). Associations with survival outcomes were studied by Cox regression models. Results: We observed an elevated CD44 and c-MET expression in metastatic cells compared to the primarily growing xenotransplantants. Moreover, we found mdm2 gene amplification was associated with tumor growth and pronounced metastatic potential in NSG mice. Functional assays unveiled a reduced viability, proliferation, and migration capacity of inherently mdm2 positive breast cancer cells upon mdm2 knock-down or anti-mdm2 targeting. Validation of mdm2 gain in luminal BC cohort within the GeparTrio trial revealed a significant association of mdm2 amplification with worse DFS (HR=1.80 [95%CI 1.16-2.79], log rank p=0.008) and OS (HR=1.75 [95%CI 1.00-3.05], log-rank p=0.047). This association was even stronger in luminal-A BC: DFS (HR=2.56 [95%CI 1.40-4.71], log rank p=0.002 and OS (HR=3.27 [95%CI 1.51-7.09], log-rank p=0.001). Conclusions: Mdm2 gene amplification facilitates ER-positive BC engraftment and progression in a preclinical in-vivo xenograft humanized NSG mouse model. Targeting mdm2 in-vitro reduced malignant cell propagation and growth. In addition, an increased mdm2 gene dose is strongly associated with an unfavorable outcome of luminal BC. Prospective studies are required to verify the suitability of mdm2 for advanced luminal BC stratification and therapeutic targeting. Citation Format: Anja Kathrin Wege, Valentina Vladimirova, Christine Solbach, Eva-Maria Rom-Jurek, Jens-Uwe Blohmer, Paul Jank, Bruno Sinn, Andreas Trumpp, Elisabetta Marangoni, Knut Engels, Wilko Weichert, Nicole Pfarr, Christoph Irlbeck, Bernhard Polzer, Olaf Ortmann, Marion van Mackelenbergh, Carsten Denkert, Sibylle Loibl, Gero Brockhoff. Mdm2 gene amplification in estrogen receptor-positive breast cancer cells is associated with enhanced solid tumor growth and pronounced metastatic potential in humanized tumor mice (HTM) and a poor outcome of patients with luminal breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD9-07.

Distinct genetic landscape and a low response to doxorubicin in a luminal-A breast cancer cell line of Pakistani origin.

Breast cancers exhibit genetic heterogeneity which causes differential responses to various chemotherapy agents. Given the unique demographic and genomic background in South Asia, genetic architecture in breast cancers is not fully explored. In this study, we determined the genetic landscape of our previously established luminal-A subtype breast cancer cell line (BC-PAK1), and compared it with a Caucasian origin MCF7 breast cancer cell line of the same molecular subtype. Deep whole-exome sequencing (100X) was performed from early passages of the primary cancer cells using the Illumina NextSeq500. Data analysis with in silico tools showed novel non-silent somatic mutations previously not described in breast cancers, including a frameshift insertion (p.Ala1591AlafsTer28) in CIC, and a frameshift deletion (p.Lys333LysfsTer21) in PABPC1. Five genes CDC27, PIK3CG, ARAP3, RAPGEF1, and EFNA3, related with cell cycle pathway (hsa04110), ErbB signaling pathway (hsa04012), Ras signaling pathway (hsa04014), and Rap1 signaling pathway (hsa04015) were found to have recurrent non-silent somatic mutations. Further, the major contribution of COSMIC signatures 3 (failure of DNA double-strand break repair by homologous recombination), and 12 (transcriptional strand-bias for T>C substitutions) was observed. Also, the somatic mutations landscape in BC-PAK1 was found to be different as compared to the MCF7 cell line. The unique genetic landscape of BC-PAK1 might be responsible for significantly reduced response to doxorubicin than the MCF7 cell line. This study presents a distinct genetic architecture in luminal-A breast cancer potentially responsible for differential response to chemotherapy. Further studies on large cohorts of breast cancer patients are suggested for implementation in personalized medicine.

Mutant p53L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to MortaparibPlus, a Multimodal Small Molecule Inhibitor.

Simple SummaryTumor suppressor protein p53 is a master regulator that inhibits the process of oncogenesis by induction of cell senescence/cell cycle arrest/apoptosis during normal and stressed states of cells. It is functionally inactivated in the majority of cancers. Mortalin, a member of the Hsp70 family of proteins, enriched in cancer cells, is known to cause cytoplasmic sequestration and inactivation of the p53’s transcriptional activation function. Inhibition of mortalin–p53 interaction and reactivation of p53 functions by natural and synthetic drugs has emerged as a possible cancer therapeutic strategy. We recently reported a novel multimodal small molecule, named MortaparibPlus, that inhibited mortalin–p53 interaction and caused reactivation of p53 function in colorectal cancer cells. Here, we report its effect on breast cancer cells with wildtype (MCF-7) or mutant (T47D) p53 status.We previously performed a drug screening to identify a potential inhibitor of mortalin–p53 interaction. In four rounds of screenings based on the shift in mortalin immunostaining pattern from perinuclear to pan-cytoplasmic and nuclear enrichment of p53, we had identified MortaparibPlus (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole) as a novel synthetic small molecule. In order to validate its activity and mechanism of action, we recruited Luminal-A breast cancer cells, MCF-7 (p53wild type) and T47D (p53L194F) and performed extensive biochemical and immunocytochemical analyses. Molecular analyses revealed that MortaparibPlus is capable of abrogating mortalin–p53 interaction in both MCF-7 and T47D cells. Intriguingly, upregulation of transcriptional activation function of p53 (as marked by upregulation of the p53 effector gene—p21WAF1—responsible for cell cycle arrest and apoptosis) was recorded only in MortaparibPlus-treated MCF-7 cells. On the other hand, MortaparibPlus-treated T47D cells exhibited hyperactivation of PARP1 (accumulation of PAR polymer and decrease in ATP levels) as a possible non-p53 tumor suppression program. However, these cells did not show full signs of either apoptosis or PAR-Thanatos. Molecular analyses attributed such a response to the inability of MortaparibPlus to disrupt the AIF–mortalin complexes; hence, AIF did not translocate to the nucleus to induce chromatinolysis and DNA degradation. These data suggested that the cancer cells possessing enriched levels of such complexes may not respond to MortaparibPlus. Taken together, we report the multimodal anticancer potential of MortaparibPlus that warrants further attention in laboratory and clinical studies.

Disease recurrence and oncological outcome of patients treated surgically with curative intent for estrogen receptor positive, lymph node negative breast cancer

BackgroundThe molecular era has identified four breast cancer subtypes. Luminal A breast cancer (LABC) is defined by estrogen-receptor positive (ER+), progesterone-receptor positive (PgR+) and human epidermal growth factor receptor-2 negative (HER2-) tumours; these cancers are the most common and carry favourable prognoses. AimsTo describe clinicopathologic features, oncological outcome and relapse patterns in LABC. MethodsConsecutive female patients diagnosed with ER/PgR+/HER2-, lymph node negative (LN-) breast cancer between 2005 and 2015 were included. Clinicopathological and recurrence data was recorded using descriptive statistics. Oncological outcome was determined using Kaplan-Meier and Cox-regression analyses. ResultsAnalysis was performed for 849 patients with median follow-up of 102.1 months. Mean disease-free (DFS) and overall survival (OS) were 85.8% and 91.8%. Seventy patients died during this study (8.2%), while 58 patients had recurrence; 7 had local recurrence (0.8%) and 51 had distant recurrence (DDR) (6.0%). Patients developing DDR were likely to be postmenopausal (P = 0.028), present symptomatically (P < 0.001) and have larger tumours (P < 0.001). The mean time to DDR was 65.7 months, with fatal recurrence occurring in 66.6% of patients with DDR (34/51). Systemic chemotherapy prescription did not influence DDR (P = 0.053). Age >65 (hazards ratio (HR):1.66, 95% Confidence Interval (CI):1.07–2.55, P = 0.022), presenting symptomatically (HR:2.28, 95%CI:1.21–4.29, P = 0.011) and tumour size >20 mm (HR:1.81, 95%CI:1.25–2.62, P = 0.002) predicted DFS, while age>65 (HR:2.60, 95%CI:1.49–4.53, P = 0.001) and being postmenopausal at diagnosis (HR:3.13, 95%CI:1.19–8.22, P = 0.020) predicted OS. ConclusionOur series demonstrated excellent survival outcomes for patients diagnosed with LN- LABC after almost a decade of follow-up. However, following DDR, fatal progression is often imminent.

Significance of Targeting VEGFR-2 and Cyclin D1 in Luminal-A Breast Cancer.

The hormonal luminal-A is the most pre-dominant sub type of breast cancer (BC), and it is associated with a high level of cyclin D1 in Saudi patients. Tamoxifen is the golden therapy for hormonal BC, but resistance of cancer cells to tamoxifen contributes to the recurrence of BC due to many reasons, including high levels of AIB1 and cyclin D1. Overcoming drug resistance could be achieved by exploring alternative targetable therapeutic pathways and new drugs or combinations. The objective of this study was to determine the differentially enriched pathways in 12 samples of Saudi women diagnosed with luminal-A using the PamChip peptide microarray-based kinase activity profiling, and to compare the activity of HAA2020 and dinaciclib with tamoxifen in singles and combinations in the MCF7 luminal-A cell line. Our results of network and pathway analysis of the 12 samples highlighted the importance of VEGFR and CDKs in promoting luminal-A breast cancer. The activation of VEGF signaling via VEGFR-2 leads to activation of PI3K/AKT kinases and an increase of cell survival, and leads to activation of Hsp90, which induces the phosphorylation of FAK1, resulting in cytoskeleton remodeling. PLC-gamma 1 is also activated, leading to FAK-2 and PKC activation. Notably, the G1/S cell cycle phases and phosphorylation processes contribute to the top seven tumorigenesis processes in the 12 samples. Further, the MTT combination of HAA2020 and dinaciclib showed the best combination index (CI), was more clonogenic against MCF7 cells compared to the other combinations, and it also showed the best selectivity index (SI) in normal MRC5 cells. Interestingly, HAA2020 and dinaciclib showed a synergistic apoptotic and G1 cell cycle effect in MCF7 cells, which was supported by their synergistic CDK2, cyclin D1, and PCNA inhibition activities. Additionally, the combination showed VEGFR-2 and Hsp90 inhibition activities in MCF7 cells. The results show the significance of targeting VEGFR-2 and cyclin D1 in Saudi luminal-A breast cancer patients, and the effect of combining HAA2020 and dinaciclib on those targets in the MCF7 model. It also warrants further preclinical and in vivo investigations for the combination of HAA2020 and dinaciclib as a possible future second-line treatment for luminal-A breast cancers.

Germline APOBEC3B deletion influences clinicopathological parameters in luminal-A breast cancer: evidences from a southern Brazilian cohort.

APOBEC3A and APOBEC3B cytidine deaminases have been implicated in the pathogenesis of multiple cancers, including breast cancer (BC). A germline deletion linking APOBEC3A and APOBEC3B loci (A3A/B) has been associated with higher APOBEC-mediated mutational burden, but its association with BC risk have been controversial. Therefore, this study investigated the association between A3A/B and BC susceptibility and clinical presentation in a Brazilian cohort. A3A/B deletion was evaluated through allele-specific PCR in 341 BC patients and 397 women without familial or personal history of neoplasia from Brazil and associations with susceptibility to BC subtypes were tested through age-adjusted logistic models while correlations with clinicopathological parameters were tested using Kendall's tests. No association was found between A3A/B and BC susceptibility; however, in Luminal-A BCs, it was positively correlated with tumor size (Tau-c = 0.125) and Ki67 (Tau-c = 0.116) and negatively correlated with lymph node metastasis (LNM) (Tau-c = -0.162). The negative association between A3A/B with LNM in Luminal-A BCs remained significant even after adjusting for tumor size and Ki67 in logistic models (OR = 0.22; p = 0.008). These results show that although A3A/B may not modify BC susceptibility in Brazilian population, it may affect clinicopathological features in BC subtypes, promoting tumor cell proliferation while being negatively associated with LNM in Luminal-A BCs.

Utility of Circulating Tumor Cells for Detection of Early-Stage Luminal A Breast Cancer

Patients with early-stage luminal A breast cancer (LABC) have better prognoses. However, follow-up examinations are frequent and remain complex. The present study examined whether circulating tumor cell (CTC) detection could be used as an earlier and more reproducible indicator of disease status among patients with early-stage LABC, and given China's healthcare resource challenges, whether it could periodically replace follow-up routine imaging. A total of 135 postoperative Chinese patients with early-stage LABC were randomly assigned to a CTC group (68 patients underwent alternating assessments using CTC detection and routine re-examination) or control group (67 patients underwent only routine re-examination). The prognosis and patient-covered costs of the various assessments were calculated for the 2 groups. No patients had normal CTCs and simultaneous abnormal imaging findings. There were no differences in overall survival, disease-free survival and total patient-covered cost of follow-up between the 2 groups (all P > 0.05). However, there was a significant difference in the average patient-covered cost (P < 0.001). Furthermore, significant intergroup differences were observed in the total and average hospitalization times (P < 0.05). Among Chinese patients with low-risk LABC, CTC detection was highly reliable and relatively low cost. Therefore, CTC detection may be used to reduce the number of routine imaging follow-ups.

Heterocellular gene signatures reveal luminal-A breast cancer heterogeneity and differential therapeutic responses

Breast cancer is a highly heterogeneous disease. Although differences between intrinsic breast cancer subtypes have been well studied, heterogeneity within each subtype, especially luminal-A cancers, requires further interrogation to personalize disease management. Here, we applied well-characterized and cancer-associated heterocellular signatures representing stem, mesenchymal, stromal, immune, and epithelial cell types to breast cancer. This analysis stratified the luminal-A breast cancer samples into five subtypes with a majority of them enriched for a subtype (stem-like) that has increased stem and stromal cell gene signatures, representing potential luminal progenitor origin. The enrichment of immune checkpoint genes and other immune cell types in two (including stem-like) of the five heterocellular subtypes of luminal-A tumors suggest their potential response to immunotherapy. These immune-enriched subtypes of luminal-A tumors (containing only estrogen receptor positive samples) showed good or intermediate prognosis along with the two other differentiated subtypes as assessed using recurrence-free and distant metastasis-free patient survival outcomes. On the other hand, a partially differentiated subtype of luminal-A breast cancer with transit-amplifying colon-crypt characteristics showed poor prognosis. Furthermore, published luminal-A subtypes associated with specific somatic copy number alterations and mutations shared similar cellular and mutational characteristics to colorectal cancer subtypes where the heterocellular signatures were derived. These heterocellular subtypes reveal transcriptome and cell-type based heterogeneity of luminal-A and other breast cancer subtypes that may be useful for additional understanding of the cancer type and potential patient stratification and personalized medicine.

Elevated plasma levels of tissue factor as a valuable diagnostic biomarker with relevant efficacy for prediction of breast cancer morbidity.

The aim of this study was to evaluate the concentrations of tissue factor (TF) and its inhibitor (TFPI), vascular endothelial growth factor A (VEGF-A), soluble forms of VEGF receptors type 1 and 2 (sVEGFR1 and aVEGFR2) in patients diagnosed with luminal A breast cancer (BrC) and in healthy individuals and to find associations of analyzed factors with demographic, clinical and pathological characteristics in a homogeneous breast cancer group. Study group consisted of 60 women aged 40 - 69 years, diagnosed with luminal-A subtype of BrC, without distant metastases (M0). Control group comprised 40 healthy women aged 45 - 63 years. Blood samples were collected from each patient in order to determine plasma levels of TF, TFPI, VEGF-A and sVEGFR1 and sVEGFR2. The examined parameters were measured by enzyme-linked immunosorbent assay (ELISA). The capacity of angiogenic and hemostatic parameters in predicting neoplasm disease was analyzed using receiver operating characteristic (ROC) curve analysis. According to ROC curve analysis, the optimum cut-off point for TF was 304.58 pg/ml, with 100% sensitivity and 100% specificity, which was calculated to discriminate between controls and malignancy patients. In luminal A BrC patients there were significantly higher concentrations of VEGF-A and TF than in controls. On the contrast the levels of sVEGF receptors type 1 and 2 as well as TFPI in luminal-A BrC cases were significantly lower in respect to healthy volunteers. Levels of examined factors in the study group varied depending on age, menopausal status, lymph node involvement and histological type. We concluded that altered levels of examined factors in patients diagnosed with luminal-A breast cancer indicate increased activation of angiogenesis and hemostasis. The results obtained may be indicative of a mutual connection between angiogenesis and hemostasis processes in tumor development and progression. Clinical and pathological parameters may possibly affect levels of angiogenic and coagulation factors.

Abstract P4-08-35: Young age at diagnosis is associated with worse prognosis in the luminal A breast cancer subtype. A retrospective institutional cohort study

Abstract Background: Breast cancer (BCA) presents with distinct molecular subtypes, each associated with different patterns of relapse, drug sensitivity, and prognosis. Although age at diagnosis is a recognized independent prognostic risk factor, its relative importance among molecular subtypes is not well documented. The aim of this study was to evaluate the prognostic role of age at diagnosis among BCA patients of different immunohistochemical subtypes (LuminalA, LuminalB, Her2, Triple-negative). Methods: We conducted a retrospective study of women with invasive BCA undergoing surgery at the Johns Hopkins Hospital from January 2000 - December 2016, excluding patients presenting with stage IV breast cancer. Patients were stratified into three age groups: ≤ 40, 41-60, and &amp;gt; 60 years, and multivariable analysis was performed using Cox regression. To explore age-related differences in gene expression, we identified differentially expressed genes (DEG) between age groups among BCA subtypes in the TCGA dataset. Finally, we identified key driver genes within DEG using a weighted gene co-expression network analysis. Results: Our cohort included 3,524 patients with a median follow-up of 85.1 months. LuminalA breast cancer patients had significantly lower 5-year Disease Free Survival (DFS) and Distant Metastasis-Free Survival (DMFS) in the ≤ 40 year age group compared to the 40-60 year age group (HR=2.69, 95%CI: 1.72 - 4.23 and HR=2.95, 95%CI: 1.78 - 4.90, respectively), while the other molecular subtypes showed no significant association of DFS or DMFS with age. Age was a stronger predictor of 5- year DFS and 5-year DMFS than tumor grade or proliferative index (Ki67) in LuminalA BCA patients, but not other subtypes. Gene expression data were obtained from 1097 BCA TCGA patients, divided into two groups (≤40y, n=36; &amp;gt;40y, n=455). We identified 374 DEG between ≤40y and &amp;gt;40y LuminalA BCA subsets. The DEG were enriched in 7 pathways, and the WGCNA analysis identified two modules of co-expressed genes. No age group-specific DEG were identified in non-LuminalA subtypes. Conclusion: Age at diagnosis may be an important prognostic factor in LuminalA BCA and may improve risk stratification and personalized therapy. Prospective studies are needed to further evaluate the prognostic value of age in this subset of BCA patients. Citation Format: Liu Z, Sahli Z, Wang Y, Wolff AC, Cope L, Umbricht CB. Young age at diagnosis is associated with worse prognosis in the luminal A breast cancer subtype. A retrospective institutional cohort study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-35.

Abstract P1-05-02: Novel mechanisms of Rac1 activation by the Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer

Abstract Overexpression of HER2 in breast cancer is correlated with poor prognosis. HER2-targeted drugs, such as trastuzumab and lapatinib, have been successful to treat HER2-positive breast cancers, however, the acquisition of the drug resistance of the cells is recognized. Here we suggest the novel molecular targets to cure HER2-positive breast cancers. The oncogenic roles of Rac1, a Rho family small GTPase, in a variety of cancers have been demonstrated. For example, the elevated expression or hyperactivation of Rac1 is frequently observed in human cancers, correlating with their aggressiveness and poor prognosis. In breast cancers, upregulation of Rac1 GEF (GTP exchange factor) and downregulation of Rac1 GAP (GTPase activating protein) have been reported. Moreover, activation of Rac1 contributed to trastuzumab resistance, which poses a serious problem during chemotherapy. In the present study, we first investigated in detail in which subtypes of breast cancers mRNA expression of Rac1 is correlated with their poor prognosis. Using the METABRIC database, we found that high mRNA expression of Rac1 significantly correlated with the poor prognosis of HER2-positive breast cancer (p=0.0012, High: n=49, Low: n=171). On the other hands, other three types (basal, claudin-low, or luminal-B type) did not show significant correlation between the expression levels of Rac1 mRNA and their prognosis (p=0.15, High: n=97, Low: n=102; p=0.052, High: n=110, Low: n=89; p=0.17, High: n=70, Low: n=391; respectively). In luminal-A type breast cancer, low mRNA expression of Rac1 significantly correlated with poor prognosis (p=0.0046, High: n=492, Low: n=187). We next investigated the molecular mechanism underlying Rac1 activation in HER2-positive breast cancer cells, SKBR-3 cells. We found that Cullin-3 (CUL3, a subunit of a RING ubiquitin E3 ligase complex) and its adaptor protein KCTD10 are essential for Rac1 activation. Mechanistically, CUL3/KCTD10 ubiquitinate RhoB, a Rho family small GTPase that suppresses the activation of Rac1, leading to the degradation RhoB. We also found that HER2 signaling is essential for the activation of Rac1. Conclusions: This study reveals that the novel molecular axis CUL3/KCTD10/RhoB regulates the Rac1 activation in HER2-positive breast cancer cells. The interference of CUL3/KCTD10 complex formation may be a new strategy to the treatment of HER2- and Rac1-positive breast cancers. Citation Format: Murakami A, Maekawa M, Kusakabe E, Yamasawa H, Aoki R, Komatsu S, Taguchi K, Nishiyama K, Yamashita M, Sugimori W, Kawai K, Nakayama J, Araki N, Semba K, Taguchi T, Kamei Y, Takada Y, Higashiyama S. Novel mechanisms of Rac1 activation by the Cullin-3/KCTD10 ubiquitin E3 complex in HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-02.

Abstract P1-13-07: Effect of adjuvant chemotherapy for patients with ER-positive/HER2-negative breast cancer assessed by the propensity score matching: Significance of nuclear grade and nodal status

Abstract Purpose: Although a survival benefit with chemotherapy (CT) is obtained over all among patients with estrogen receptor-positive (ER+) breast cancer, the degree of benefit differs among the subtypes. The 21-gene Recurrence Score assay is a validated prognostic/predictive tool for early ER+ breast cancer. However, genomic assays have not been approved in Japan and most other countries, except in some developed countries. Based on the St. Gallen 2015, 1-3 positive nodes are not an absolute indication for CT among patients with ER+/HER2-negative breast cancer. In contrast, it is better for CT to be performed except for low-risk groups, based on genomic tests according to the St. Gallen 2017. In fact, no prospective study has proven the survival benefit of CT specifically for luminal-A breast cancer. Therefore, we analyzed the survival benefit of CT for ER+/HER2-negative breast cancer by propensity score matching (PSM). Patients and methods: Between 2000 and 2015, 895patients with stage I-III ER+/HER2-negative breast cancer who had underwent surgery in our hospital were examined. Patients with bilateral breast cancer, ER&amp;lt;10%, and preoperative treatment were excluded. The primary end point was the 5-year recurrence-free survival (RFS) and overall survival (OS) in patients matched by propensity score that estimated by a logistic regression model that included factors likely to influence the decision of whether or not to administer CT (tumor size, nuclear grade [NG], progesterone receptor status and nodal status). Results: In the entire cohort, the median age was 59 (28-95) years; 223 patients (24.9%) were node positive and 126 patients (14.1%)had NG3 disease. Overall, all patients received endocrine therapy, and 24.1% received additional CT. After a median follow-up of 68.8 months, the 5-year RFS rate was 94.3% in CT-untreated patients (non-CT group) and 90.1% in CT-treated patients (CT group; hazard ratio [HR] for recurrence, 1.47; 95% confidence interval [CI], 0.90 -2.33; p=0.106). The 5-year OS rate was 97.5% in the non-CT group and 95.6% in the CT group (HR for death, 1.80; 95% CI, 0.99-3.21; p=0.047). Using PSM, 236 patients were selected (1:1 matching between non-CT and CT groups). After matching, the 5-year RFS rate was higher in the CT group than in the non-CT group (96.8% vs. 82.7%; HR for recurrence, 0.29; 95% CI, 0.11-0.68; p=0.003), and the 5-year OS rate was higher in the CT group than in the non-CT group (100% vs. 91.9%; HR for death, 0.06; 95% CI, 0.003-0.35; p&amp;lt;0.001). Among PSM patients, with node-negative/NG3 and 1-3 node positive/NG2 disease, the 5-year RFS rate was significantly higher in the CT group than in the non-CT group (p=0.041 and p=0.006, respectively). Conclusion: No significant benefit of CT was observed when considering the entire cohort because of the treatment bias. When clinical and tumor features were matched by propensity score, the addition of CT significantly improved both RFS and OS of ER+/HER2-negative breast cancer patients, especially for patients with node- negative/NG3 and 1-3 node positive/NG2 disease. Citation Format: Tanaka N, Ogura K, Kamimura M, Hattori A, Inoue H, Yukawa H, Sakaguchi S, Matsuoka A, Kodera A, Hirano A. Effect of adjuvant chemotherapy for patients with ER-positive/HER2-negative breast cancer assessed by the propensity score matching: Significance of nuclear grade and nodal status [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-13-07.